One promising approach to cure AIDS is by gene therapy using hematopoietic stem cells (HSCs) containing transgenes that inhibit HIV replication. HSCs are promising targets for gene therapy of AIDS as they produce all of the cells involved in HIV pathogenesis; T lymphocytes, macrophages, dendritic cells and microglial cells. However, the inability to efficiently deliver anti-HIV transgenes to HSCs has been a roadblock in clinical trials. We propose to develop foamy retroviral vectors that inhibit HIV replication towards a HSC gene therapy for AIDS. Foamy vectors may be well suited for gene therapy of AIDS as they can efficiently and stably transduce HSCs. Foamy vectors expressing short hairpin RNAs (shRNAs) targeted to tat/rev, a ribozyme targeted to CCR5, a TAR decoy, and a peptide that inhibits HIV entry will be evaluated for their ability to deliver stable and specific inhibition of HIV replication to CD34-derived macrophages. Experiments will be performed to confirm that HIV vRNAs are specifically reduced in transduced HIV-resistant cells and potential toxicity from shRNA expression will be evaluated. We will determine if the colony-forming ability of transduced hematopoietic progenitors is altered or if shRNA expression affects host gene expression in HIV-resistant macrophages. The non-obese diabetic/severe combined immunodeficient NOD/SCID xenotransplantation model will be used to evaluate whether HIV resistance is maintained in human macrophages and T lymphocytes differentiated in vivo and determine if we can increase the numbers of functional HIV-resistant repopulating cells by in vivo selection. Finally in these NOD/SCID experiments we will also determine foamy vector integration sites in human repopulating cells which is a critical issue currently facing retroviral gene therapy. The proposed experiments are designed to systematically develop safe and effective foamy shRNA vectors for future studies where we assess their efficacy in primate models and eventually in clinical trials. [unreadable] [unreadable] [unreadable]